Study On Genes Related To Triterpenoid Saponn Biosynthetic Pathway In Aralia Elata

Aralia elate ( Miq. ) Seem is deciduous small arbors of Aralia Linn. in Araliaceae. Its root bark, stem bark, leaves and tender bud can be used as traditional herbal medicines. The main medicial composition is oleanane. It is hepatoprotective. But the wild resources cannot completely supply with clinical needs and the period of cultivation is a very long time. With the propose and application in China, "Chinese Genome Project", Genetic engineering shows a great prospect on resources of medicinal plant. Therefore, it is very necessary to improve the content of secondary metabolites in Aralia elate by biotechnology.The key gene of triterpenoid saponin biosynthesis pathway——Squalene synthase was cloned. Full length is 1245bp and it encodes 414 amino acids. Accession No.is GU354313. Bioinformatics analysis suggested the sequence have full open reading frame. The SS proteins from Aralia elata, Panax ginseng, Panax quinquefolius, Panax notoginseng, Bupleurum chinense, Glycyrrhiza uralensis and Arabidopsis thaliana showed 95%,91%,96%,83%,89% and 72% identities based on amino acid sequences alignment. And phylogenetic trees of gene SS was constructed.It is hydrophobic membrane-bound protein. We constructed the prokaryotic expression vector pET-28a-AeSS. SDS-PAGE and Western blot analysis suggested gene SS is expressed successfully.We constructed eukaryotes expression vector pBI121-AeSS. The tobacco was infected by leaf disc transformation. Molecular detection proved that gene SS was successfully integrated into the tobacco genome DNA. And we also constructed yeast expression vector pPIC9K-AeSS. The yeast was infected by electroporation methods.. SDS-PAGE analysis gene SS is expressed successfully.We selected root,stem,leaf and designed more than 60 culture medium to induce the formation of callus. We obtained four good culture mediums. And the content of oleanolic acid of lateral root,stem,leaf and callus were determined by HPLC. The result is that the most content is oleanolic acid in callus. The content of oleanolic acid is similar in stem and leaf. The lateral root is in the least level. We study the expression levels of gene SS in lateral root,stem,leaf and flower by Real-time PCR. The result is that the expression levels of gene SS in stem is the most, secondly leaf, lateral root is the least. The result of HPLC and Real-time RT-PCR suggests: expression differences of different organs have some effect on the content of oleanolic acid of these organs.